We have focused our studies in a number of different areas in FY2016: 1. For several years we have been interested in the role of cell surface and secreted TGF-beta in Treg Function. TGF-beta can have pleotropic effects on different cell types ranging from immune suppression, the promotion of fibrosis, and the promotion/suppression of tumor growth. Activated Treks, but not activated T conventional (Tconv) cells, express the leucine rich repeat protein, GARP, which is responsible for surface localization of latent TGF-beta1. Although TGF-beta1 has been implicated in the suppressor function of Tregs, Treg conditional knock outs of TGF-beta1 or GARP display normal suppressor function in vitro. Cell surface associated TGF-beta1 on Treg cells may also mediate immunoregulatory functions. We postulated that one cell intrinsic role of the GARP/L-TGF-beta1 complex may be to provide active TGF-beta1 for the induction of pTregs during the induction of oral tolerance. We demonstrated in an adoptive transfer model that recipient Tregs play a non-redundant role in the induction of Foxp3+ pTreg following the oral administration of antigen. This result is in contrast to the view that CD103+ DCs are the source of TGF-beta1. The contribution of recipient Treg to pTreg induction was mediated by TGF-beta1 derived from the GARP/L-TGF-beta1 complex on the recipient Tregs, as pTreg induction was impaired when the recipient Treg cells could not produce TGF-beta1 or failed to express GARP. The induction of oral tolerance was normal in animals whose Treg were deficient in integrin beta8. It is therefore likely that TGF-beta1 derived from the GARP/L-TGF-1 complex is activated by integrin beta8 expressed by CD103+ DC. 2. In general, all the proposed mechanisms for Treg function postulate that once Tregs are activated via the TCR, the suppressor-effector mechanism they utilize is non-specific. We addressed this question by asking whether iTregs specific for one antigen could suppress the activation/expansion of naive T cells specific for a distinct antigen when both were simultaneously expressed by the same DCs. iTregs specific for one antigen suppressed the activation of naive T cells specific for their cognate antigen, but had no effect on the activation of naive T cells specific for a different antigen expressed on the same DC. We therefore explored alternative mechanisms by which iTregs might mediate suppressor function. Analysis of iTreg-DC co-cultures in vitro using flow cytometry and confocal microscopy demonstrated that specific peptide-MHCII complexes were captured, expressed on the cell surface, and internalized by iTregs leaving DCs with decreased levels of antigen. Polyclonal iTregs, naive, and activated antigen-specific Teff cells did not capture peptide MHCII complexes. These studies suggest that antigen-specific iTreg inhibit immune responses locally in an antigen-specific fashion by forming firm interactions with DCs leading to a stripping of peptide-MHCII complexes from the DC.